Mammalian heart tissues contain two types of NADP-specific isocitrate dehydrogenases, one mitochondrial and the other cytoplasmic, as well as an allosteric NAD-dependent isocitrate dehydrogenase located in the mitochondria. Isocitrate dehydrogenase catalyzes one of the rate limiting steps in the energy-producing Citric Acid Cycle. Evidence suggests that, in heart failure, decreases in the NAD enzyme are associated with decreased oxidative metabolism and energy production. In contrast, the NADP-specific isocitrate dehydrogenases have a major role in the generation of NADPH for reductive biosynthesis and protection against oxidative stress in the heart. Our goal is to understand the structural basis for differences in kinetics, specificity and regulation of the two mammalian mitochondrial isocitrate dehydrogenases. We hypothesize that certain critical amino acids involved in catalysis and metal-isocitrate binding are conserved among the isocitrate dehydrogenases, but that there is greater diversity at the coenzyme and nucleotide regulatory sites. The recombinant pig heart mitochondrial NADP-specific isocitrate dehydrogenase is a dimer of identical subunits and its activity is not allosterically regulated. We aim to identify its critical amino acids by site-directed mutagenesis, affinity labeling and x-ray crystallography. Target sites for mutation will be chosen on the basis of sequence alignments among the isocitrate dehydrogenases, affinity labeling results, and analysis of crystal structures. Mutant enzymes will be purified and extensively characterized. We have crystals (diffracting to 1.7 A) of the Mn-isocitrate complex of this recombinant wild type NADP enzyme. We now propose to solve its structure and to determine that of other wild type enzyme-ligand complexes, as well as those of selected mutant enzymes.The mammalian NAD-specific enzyme is activated by ADP and has 3 types of subunits present in the ratio 2alpha: 1beta: lgamma. The subunits of the human enzyme have recently been co-expressed in E. coli. With the aim of elucidating the roles of these subunits, we plan to express, purify, characterize and compare the NAD enzyme composed of wild type alpha, beta and gamma subunits, with enzyme composed of 2 wild type subunits + 1 subunit in which a single amino acid (proposed to participate in catalysis, substrate or ADP binding) is replaced by mutagenesis. Knowledge of the isocitrate dehydrogenases at the molecular level is important for understanding the role of these enzymes in human cardiac energy metabolism, and in the cellular defense against damage caused by reactive oxygen species. These studies may lead to the rational design of synthetic activators of isocitratedehydrogenases useful for treating human disease.